免疫荧光的方法依据你使用的试剂盒各有不同，最好你仔细阅读试剂盒的说明书按照所给程序进行操作。+ P* M0 G- y2 H0 r# x- o
给你一个方法参考一下，不过是写的荧光双标，与单标的过程基本是类似的只是双标多标了一次。' E) E8 A/ ?. B; m Y S
Double immunofluorescence staining for BCL-6 and else.% B! C7 U5 m& a
Any type of tissue is suitable for this technique, as long as the antigenicity for your antigen(s) is preserved. $ n6 ?, G5 o6 B
Use cytocentrifuged cells or frozen or paraffin dewaxed sections. / q- n L0 G& b/ F1 K3 ~! E' DPresence of endogenous fluorescent substances may affect your staining. ) u4 g* r: q4 ^
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Note: This protocol uses a twice repeated sequence of primary and secondary layers in order to amplify the signal, as in the PAP / APAAP technique. ) c- J- I+ W' g3 JStart at this section for unfixed or acetone-fixed specimens. $ p. L# Q. X) c4 w" Y6 x' a
" X8 i( p7 y4 N' l, s1- Mark the slide in order to recognize the area to be stained and label the slide. % |3 L% D4 V) W8 `( D* ^2- Fix in acetone for 10 min at RT [this tep can be omitted]. + t: B( e0 |6 ^
3- Blot the slides and let them dry for 10 min - 2 hrs. 0 D7 p' S8 |4 n1 |6 H% G4- Fix in 10% formalin for 10 min in a Coplin Jar. : c( D# {4 R. l' ~2 e2 k0 c' xFrom this point on, don't let dry the slides at any time. 7 F) N% m: S1 M* P1 O5 v! j, u
5- Discard the formalin appropriately and quickly wash a few times in distilled water (from the distilled tap is OK). 0 P% [# X y1 c: f( B& _- |8 y* V2 S/ v: p$ w
Start at this section for dewaxed, antigen retrieved slides 2 W8 h: z, l! c$ p8 g$ |
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6- Wash twice in TBS 0.05M pH7.5, to which 0.01% Tween 20 has been added. 5 minutes / wash. 1 ?3 \* k; [" ^. z- K% F7- Briefly blot the slides without letting them dry and then apply 3% human serum (health hazard!) or pig serum a s a blocking agent., J0 P+ P* A% \$ [
8- incubate with the blocking for 10 min. 1 w$ U z m& C M8 [
9- blot the slides without washing and apply a mixture of the primary antibodies, in a moist chamber, at RT for 1-18 hr. 1 ~6 ?5 b7 |) K" x4 s
10- Wash twice. 4 X$ L8 j& S& ~5 @( k0 K11- apply a mixture of secondary antibodies (see example below) for 45 min at RT. ) {* G- o F% q( A s8 l12- Wash twice. 5 S1 x6 r4 b/ M1 D9 s( P0 r
13- add the mixture of primary antibodies and incubate for 15 min. $ Z% C; v( C$ _: ]# z
14- Wash twice . 3 W. j& i- g! F0 R6 V
15- add the mixture of secondary antibodies and incubate for 15 min. ( G: \) o$ k9 S4 Q( W- c9 |( _4 b( k7 `' a P2 Q8 u
proceed with steps 16 through 19 if one of the secondary is biotin-conjugated. Else go to 19.5 j0 a+ `# D1 O
5 r3 {! x9 t q* D) M& K16- dilute the avidin-fluorochrome in TBS/Tween and centrifuge for 15 min. / [* s" M2 h% Q# W) t$ r17- Wash thrice. . J# @8 g x: P' D0 A
18- Incubate with the avidin for 15 min. ; W0 I6 j7 Z4 C7 ~19- Wash thrice in TBS 0.05M pH7.5 + 0.01% Tween 20 and mount.5 g5 T8 E* p8 Q
K6 ^8 W& J$ dExample: Pirmary antibodies: rabbit anti-BCL-6 0.5µg/ml in PBS/BSA/Azide + mouse anti HA supn 1:100. % U% P8 \6 d2 G2 p, i; g
Secondary antibodies: biotin-conjugated goat anti rabbit (1:200) + TRITC conjugated goat anti-mouse (1:200). Alternatively, biotin-conjugated goat anti mouse (1:200) + goat anti rabbit TRITC (1:200) 0 K: p0 T, s0 v8 u: v2 \3 _
Avidin FITC: 1:300 " M8 B9 B) ~7 D- v y7 f/ x
To see the examples click on the thumbnail. , J# m) k+ K( A ' v$ O6 Y- r: F" i* I! o. T $ u0 U ^; f1 g) Z/ x$ [
8 q# w, n) S8 _ 作者: 骨道边 时间: 2011-6-9 12:03